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Necrostatin-1 reverts shikonin-induced necroptosis to apoptosis

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Abstract

Degterev et al. previously demonstrated that death receptor mediated apoptosis could be diverted to necroptosis when apoptosis signaling was blocked, suggesting that necroptosis may function as a backup mechanism to insure the elimination of damaged cells under certain conditions when apoptosis was inhibited. Here, we show that shikonin-induced necroptosis can be reverted to apoptosis in the presence of necrostatin-1 (Nec-1), a specific necroptosis inhibitor and that the death mode switch is at least partially due to the conversion from mitochondrial inner membrane permeability to mitochondrial outer membrane permeability, which is associated with Bax translocation. The data combined with the previous reports support a notion that apoptosis and necroptosis may function as reciprocal backup mechanisms of cellular demise. To the best of our knowledge, this is the first study to document a conversion from necroptosis to apoptosis.

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Abbreviations

DR:

Death receptor

Nec-1:

Necrostatin-1

SHK:

Shikonin

MIMP:

Mitochondrial inner membrane permeability

MOMP:

Mitochondrial outer membrane permeability

ROS:

Reactive oxygen species

RIP1:

Receptor-interacting protein 1

P-gp:

P-glycoprotein

MRP1:

Multidrug-resistance associated protein 1

PT pore:

Permeability transition pore

∆ψm :

Mitochondrial trans-inner-membrane potential

MMP:

Mitochondrial membrane potential

VP16:

Etoposide

STS:

Staurosporine

CsA:

Cyclosporin A

BA:

Bongkrekic acid

DIDS:

4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate

MTR:

Mitotracker red

JC-1:

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-benzimidazo-lylcarbocyanine iodide

Ro 5-4864:

4′-Chlorodiazepam

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Acknowledgments

This work was supported in part by Cheung Kong Scholars Programme (National Ministry of Education, China, and Li Ka Shing Foundation, Hong Kong), China National 863 project (2007AA02Z143), China National Ministry of Health Grant (WKJ2006-2-11), the Bureau for Traditional Chinese Medicine Grant 2006Z013A, Zhejiang Province, China (all to XH).

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Correspondence to Xun Hu.

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10495_2009_334_MOESM1_ESM.tif

Supplementary Fig. 1 Death mode induced by shikonin is dose-dependent HL60 cells were treated with shikonin at various concentrations for 12 h, nuclear morphology was detected by Hoechst 33342 staining.

10495_2009_334_MOESM2_ESM.tif

Supplementary Fig. 2 Necrostatin-1 converts shikonin-induced necroptosis to apoptosis in K562 or K562/Adr A, Cells were treated with shikonin at 10 μM in the presence or absence of Nec-1 for 12 h, and apoptotic or necrotic death rates was counted by Vital dye exclusion assay and Hoechst-staining. B & C, Activation of caspases 3, 8 and 9 in K562 and K562/Adr cells treated with shikonin for 6 h in absence or presence of Nec-1. Data are mean ±SD or representative of at least 3 independent experiments.

10495_2009_334_MOESM3_ESM.tif

Supplementary Fig. 3 Plasma membrane of apoptotic cells is not necessarily permeable to trypan blue HL60 Cells were treated with 2.5 μM shikonin for 12 h. Apoptotic or necrotic death rates was counted by vital dye exclusion assay and Hoechst-staining. Data are mean ± SD.

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Han, W., Xie, J., Li, L. et al. Necrostatin-1 reverts shikonin-induced necroptosis to apoptosis. Apoptosis 14, 674–686 (2009). https://doi.org/10.1007/s10495-009-0334-x

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